Indian Journal of Fibre & Textile Research
Vol. 25, March 2000, pp. 69-74
Degumming of silk with lipase and protease
M L Gulrajani & Ritu Agarwal
Department of Textile Technology, Indian Institute of Technology, New Delhi 110016, India
and
Amrit Grover & Mona Suri
Lady Irwin College, New Delhi 11000 I, Indi a
Received J8 Jam'([lI), J999; accepted J3 April J999
A lipase enzyme in combinati on with a protease enzyme has been used for dewaxing and degumming of silk and
the combined effect of dewaxing and degumming on weight loss, wettability , dye uptake, yellowing, microscopic
struc ture, handle and lustre has been studied. It is observed that the combined enzyme treated samples show the
same we ight lo ss, cleaner longitudinal su rface and better wettability when compared with the Marseill es' soap
treated samples .
Keywords: Dewaxing, Degumming, Lipase, Protease, Silk
1 Introduction
The raw si lk does not merely consist of an inner
filament fibroin , sheathed in natural size sericin , but
is also contaminated with small amounts of nonproteinaceous impurities like du st, minerals, pigments
and waxy matter'. Along with sericin, these waxes
playa role in obscuring the brilliance and softness of
the silk . Only a few studi es have been conducted on
waxes and oils present in si lk, their nature, composition, properties, exact quantities and method s of removal. Apart from naturall y occurring waxes and
oils, some additional waxes are applied to the yarns
while weaving them into fabric which do not get removed even after degumming. These waxes hamper
the processi ng of si lk due to poor wetting properties.
Hence, complete elimi nation of these waxy substances is very essential. Due to the ambi guity about
the true nature and composition of these waxes, no
method has been successfully de vised to remove them
completely. It is believed that a fraction of these
waxes is saponifiabl e and therefore can be removed
with alkali or soap treatment, but these method s are
believed to be harmful to fibroin . The use of enzymes
in the silk indu stry is relatively unexplored and has
generated a lot of interest and much research is being
carried out internationally. For this purpose, a special
class of enzymes called lipases, which are specialised
in hydrolysing waxes into fatty acids and alcohols,
seem to be aR appropriate answer.
The present work was therefore undertaken to
study the effect of lipase treatment on silk wax and
the combined effect of dewaxing and degumming on
weight loss, wettability, dye uptake, microscopic
structure, handle and lustre.
2 Materials and Methods
2.1 Materials
Two varieties of raw si lk fabric with plain weave
(S ilk A) and crepe weave (S ilk B) were used in thi s
study.
Lip ase enzyme Amano AK 20, from Pseudomonas sp., with activity of 9.98 AU/g wa s
obtained from th e Department of Biochemical
Engineering a nd Bi otec hn ology, lIT-Delhi. Protease enzyme Alcalase 2,4 LFG with activity of
2.4 AU/g was procured from Novonordisk ,
Denmark .
2.2 Methods
2.2.1 Preparation of Samples
Samples from each silk were cut weighing 3 g
each. They were conditioned at 30% RH and 25°C for
48 h and weighed again.
2.2.2 Fabric Treatment
The recipes used for treatment of the fabric with
lipase, protease and soap are given below:
INDIAN 1. FIBRE TEXT. RES., MARCH 2000
70
Dewaxing (with lipase)
Buffer solution
Lipase
Temperature
pH
Time
MLR
Degumming (with protease)
Alcalase
Sandopan DTC
NaHCO J
Temperature
pH
Time
MLR
O. IM NaHC0 3
10% owf
40%C
8-8.5
2h
1:50
10% owf
1.5 gpl
7.5% owf
60°C
8.5
2h
1:50
Degumming (with Marseilles ' soap)
Marseilles' soap
25 % owf
Temperature
93°C
pH
10
Time
1.5 h
MLR
1:50
2.2.3 Optimization of Lipase Concentration
The samples were treated with 0%, 5% and 10%
lipase enzyme . The effectiveness of lipase was estimated by measuring the weight loss .
2.2.4 Optimization of Conditions for Alcalase Treatment
Degummi ng of dewaxed and raw silk samples was
carried out with two concentrations of Alcalase, viz.
10% and 15 % (owf), for 45 min and 3 h.
2.2.5 Combined Treatment of Lipase and Alcalase
To see the effect of combined treatment of lipase
and protease in the same bath the samples were
treated under the following conditions :
Alcalase
10% owf
Lipase
10% owf
Sandopan DTC
1.5 gpl
0.1 M NaHC0 3
Buffer solution
Temperature
60°C
pH
8.5
Time
2h
MLR
1:50
2.2.6 Determination of Weight Loss
Samples were weighed accurately before and after
the enzyme treatment using Sartorius moisture analyzer and the weight loss was calculated as follow s:
Weight loss (%)
= (WI WI
W2 )
x 100
where WI and W2 are the weights of fabric before and
after the enzyme treatment.
2.2.7 Determination of Wettability
Five samples (I in.x3 in .) were cut fro m both warp
and weft directions. On each sample, two lines were
drawn I inch apart with an ink marker. With the help
of a clamp stand the samples were immersed in distilled water till the lower line. The spreading of colour showed the rise of water. The time taken by water
to travel from lower mark to upper mark was recorded.
2.2.8 Determination of Increase in Dye Uptake
To see the extent of wax removal from silk, the
ease by which the sample will get dyed was measured. Samples were dyed with reacti ve dye (Remazol
Red BS ill, 3% owf) for 5, IS and 45 min at 80°C
with 60 gpl Glauber's salt, maintaining the materialto-liquor ratio at I :50. Dye uptake was measured by
taking the K/S values of the samples on the spectrophotometer.
2.2.9 Determination of Yellowing
Samples were cut in 2 in.x4 in . size and their initial whiteness was determined by taking Hunter' s
wh iteness index using spectrophotometer. The samples were than exposed to light equ ivalent to sunlight
for 24 h. Their whiteness was again measured.
2.2.10 SEM Studies
The extent of degumming can be qualitatively assessed by viewing the degummed fibres under Cambridge scanning electron microscope. Residual sericin
appears as lumps on the surface of the fibre . The extent of fibre damaged can also be assessed by this
method . The splitting of fibres into minute fibrillae is
freq uently observed in silk. This has been attributed
to the mechanical injury of the fibre caused by
chaffing and abrasion during degumming l.
2.2.11 Determination of Handle and Lustre
The samples of equal size (2 in.x2 in.) were
mounted on black sheet and ten people were asked to
evaluate these samples on the basis of handle and
lustre. Mean ranking was taken.
3 Results and Discussion
3.1 Optimization of Lipase Concentration
Treatment of Silk A with 5% owf enzyme concentration gave a weight loss of 1.37% and the control
GULRAJANI et af.: DEGUMMING OF SILK WITH LIPASE AND PROTEASE
sample gave a weight loss of 1.33%. Whereas the
treatment with 10% enzyme concentration gave a
weight loss of 4.62%. As the weight loss with 5%
enzyme concentration was very close to that of the
control sample, 10% owf enzyme concentration was
chosen. Further, the results were not reproducible.
Therefore, lipase treatmf'nt was tried as pretreatment
for degumming process.
3.2 Optimization of AJcalase Concentration
Due to the erratic weight loss, the conditions for
dewaxing treatment could [lOt be standardized. There:
fore, the conditions for the degumming treatment
which could be applied to the lipase pretreated samples were optimized. Table I shows the weight loss
obtained after the treatment of silk with AIcalase. As
the weight loss obtained after 45 min and 180 min
was comparable, time period of 45 min was taken.
Weight loss obtained after dewaxing was compared
with the weight loss obtained without dewaxing
treatment. Table 2 shows that the lipase pretreated
samples show only marginal increase in weight loss
after degumming. Therefore, lipase was not used as
pretreatment.
3.3 Effect of Combined Dewaxing and Degumming
combined treatment with aIcalase and lipase. This
shows that there is improved degumming by the combined treatment and hence improvement in absorbency. Both the silk samples show better wicking
property in warp direction due to formation of capillaries .
3.3.3 Dye Uptake
Table 5 shows that the KlS values of the combined
enzyme treated samples are comparable to that of the
samples treated with Marseilles' soap.
3.3.4 Microscopic Structure
The scanning electron micrographs of control sample, aIcalase-treated sample, lipase-treated sample,
combined enzyme treated sample and soap-treated
sample of Silk A and Silk B are shown in Figs 1 and 2
respectively. It is observed that aIcalase-treated samples are equally clean as the samples obtained on
combined enzyme treatment. But in case of only alcalase enzyme treatment, certain small remnants of
sericin are noticed. Marseilles' soap treated samples
show fibrillation of fibre. There is not much difference in Silk A and Silk B samples. In case of plain
Table 2-Weight loss obtained after seq uential treatment of li pase
and alcalase
3.3.1 Weight Loss
Table 3 shows that the maximum weight loss is
obtained by Marseilles' soap treatment and the weight
loss on combined treatment of lipase and protease is
comparable with that of soap treatment. Though the
Marseilles' soap treatment results in complete removal of serecin, the quantity of soap required is high
(25% owf) which is not economical. Al so, the high
temperature(at boil) and alkaline pH (10.5) used
might cause degradation of fibroin . The enzymatic
treatment, on the other hand, is milder (temp. 600 e
and pH 9) and gives fairly good weight loss .
3.3.2 Wettability
It is observed from Table 4 that for both Silk A
and Silk B, the minimum wicking time is in case of
Lipase conc.
(owf) %
A1calase conc.
(owf) %
Weight loss
%
10
10
10
15
10
15
17.98
19. 54
18.21
20.33
Table 3-Effect of enzyme treatment on weight loss
Treatment
10
10
15
15
45
180
45
180
Weight loss, %
Plain silk
Crepe si lk
17.98
17.68
19.59
18.50
15.45
16.84
16.49
16.30
2. 30
4.09
20.40
21.50
21 .83
2.44
5.73
19. 11
20.57
23 .55
Table 4-Effect of enzyme treatment on wicking time
Table I-Weight loss obtained after treatment with A1calase
Time
min
Weight loss, %
Plain silk
Crepe silk
Only buffer
Only lipase
Only A1calase
Lipase and A1calase
Marseilles' soap
Treatment
Alcalase
(owf), %
71
Only buffer
Only lipase
Only A1calase
Lipase and Alcalase
Marseilles' soap
Wicking time, min
Plain sil k
Crepe silk
WarpWeftWarpWeftwise
wise
wise
wise
30.0+
30.0+
1.38
1.39
6.15
30.0+
30.0+
1.23
1.98
5.93
30.0+
30.0+
1.10
0.90
4.20
30.0+
30.0+
3.80
3. 10
15.0+
INDIAN 1. FIBRE TEXT. RES., MARCH 2000
72
Table 5-Effect of enzy me treatment on K/S value
K/S value
Treatment
Only Alcalase
Lipase and Alcalase
Marseilles' soap
5 min
Plain silk
15 min
45 mi n
5 min
Crepe silk
15 min
45 min
1.6827
1. 6803
4.07 10
3. 162 1
3.2209
6. 168 1
6.8706
6.7543
8.5500
1.1 294
1.175 1
1.51 92
2.5440
2.6 195
3. 5905
7.1161
7.1554
8.919 1
Fig. I-S cannin g e lectron micrographs of Silk A samples : (a) untreated , (b) treated with buffer or co ntrol , (c) treated with lipase, Cd)
treated with Alcalase, (e) treated with Alcalase and lipase, and (f) treated with Marseilles ' soap
GULRAJANI e/ al.: DEGUMMING OF SILK WITH LIPASE AND PROTEASE
Fig. 2-Seallning c1eetrOl , mi crogrnphs of Silk B samples: (n) untreated, (b) treated with buffer o r co ntrol , (c) treated with
(d) treated with Alcalase, (e) tn:ated with Alca lase 3nd lipase, and (f) ueated with Marsei ll es' soap
Table 6-Effect of enzyme treatment on handl e and lu stre
Treatment
Only buffer
Only lipase
Only Alcal ase
Lipase and Alealase
Marseilles' soap
Pl ain silk
Handl e
Lu stre
5
4
3
2
5
4
I
2
3
Crepe silk
Handie
Lustre
5
4
2-3
2-3
5
4
2
3
73
li p 3 ~e,
woven silk samp les (Silk A), more crimp is observed
than in case of crepe silk or satin weave samples
(Silk B) .
3.3.5 Haudle and Lustre
The data in Table 6 shows that Marseilles' soap treated
sample has been ranked first in case of handle and third in
case of lustre. Good handle could be due to uniform dissolution of sericin by the action of alkali, resulting in a
74
INDIAN J. FIBRE TEXT. RES ., MARCH 2000
smooth surface. Excess of alkali of the hydrolyzed soap
2
diminishes the lustre . Best results in terms of lustre is observed for the samples treated with only alcaIase. The
combined treatment gives better results in terms of lustre
but the samples are ranked lower in terms of handle as
compared to the soap-treated samples.
4 Conclusion
Lipase enzyme can be used along with protease to
improve the degumming of silk. The combined enzyme treatment gives the same weight loss, better
wettability and cleaner longitudinal surface as compared to Marseilles' soap treatment. This combined
enzyme treatment can be a useful replacement to the
traditional method of treatment with Marseilles' soap.
Acknowledgement
The authors acknowledge the financial support received from the Department of Biotechnology, Ministry of Science and technology, Govt. of India, for
conducting this study.
References
I
Gulrajani M L, Degumming of silk in Chemical processing
of silk, edited by M L Gulrajani (Indian
Institute of Technology, New Delhi), 1993,63.
2 Tsunkaye V D, J Soc Dyers Colour, 48 ( 1932) 280.